Two of the RFA main goals are the characterization of epitopes derived from the Large Scale Allergen Epitope Identification Contracts, 1) as a function of seasonality and 2) disease progression. Accordingly, we propose to test the hypothesis that different stages (in-season versus out-of-season), types (rhinitis versus asthma) and severities of allergic disease are associated with not only differential magnitude of T cell responses, but also distinctive patterns in terms of the TH subsets involved and their interplay. We will focus on immunodominant regions from three important allergens, namely timothy grass (TG), house dust mite (HDM) and cat dander (CD). In our first Aim, we propose to characterize responses in moderate/severe (MO/SA) and mild asthmatics (MA), and contrast them with those observed in non-asthmatic individuals suffering from allergic rhinitis (AR). Preliminary data suggest that differential ILIO, ILI7 and IFNy production correlate with different disease states. Here, we propose to characterize the subsets of TH cells producing these lymphokines ex vivo and following in vitro stimulation, by ELISPOT, intracellular cytokine staining (ICS) and tetramer staining assays. We will further examine whether the different subsets are distinct or can originate from each other in vitro, because of TH cell plasticity. Preliminary data also suggest that the relative balance of TH subsets differs substantially when TG and HDM responses are compared. We will therefore investigate whether different distributions of TH subsets correlate with allergen source. Our preliminary data further highlights dramatic differences in magnitude of TG responses as a function of seasonal exposure. In the second Aim we propose to determine in longitudinal studies response magnitude and TH functional subsets. In a further series of experiments we will perform challenge studies utilizing TG pollen extracts. We expect that because of a more vigorous response TH cells might be more easily detectable ex vivo, and characterized in greater detail without potential alterations introduced by in vitro culture. These studies will characterize the functional T cell subsets involved in different disease settings and as a function of seasonality.